Effect of Ku80 deficiency on mutation frequencies and spectra at a LacZ reporter locus in mouse tissues and cells.
Non-homologous end joining (NHEJ) is thought to be an important mechanism for preventing the adverse effects of DNA double strand breaks (DSBs) and its absence has been associated with premature aging. To investigate the effect of inactivated NHEJ on spontaneous mutation frequencies and spectra in vivo and in cultured cells, we crossed a Ku80-deficient mouse with mice harboring a lacZ-plasmid-based mutation reporter. We analyzed various organs and tissues, as well as cultured embryonic fibroblasts, for mutations at the lacZ locus. When comparing mutant with wild-type mice, we observed a significantly higher number of genome rearrangements in liver and spleen and a significantly lower number of point mutations in liver and brain. The reduced point mutation frequency was not due to a decrease in small deletion mutations thought to be a hallmark of NHEJ, but could be a consequence of increased cellular responses to unrepaired DSBs. Indeed, we found a substantial increase in persistent 53BP1 and gammaH2AX DNA damage foci in Ku80-/- as compared to wild-type liver. Treatment of cultured Ku80-deficient or wild-type embryonic fibroblasts, either proliferating or quiescent, with hydrogen peroxide or bleomycin showed no differences in the number or type of induced genome rearrangements. However, after such treatment, Ku80-deficient cells did show an increased number of persistent DNA damage foci. These results indicate that Ku80-dependent repair of DNA damage is predominantly error-free with the effect of alternative more error-prone pathways creating genome rearrangements only detectable after extended periods of time, i.e., in young adult animals. The observed premature aging likely results from a combination of increased cellular senescence and an increased load of stable, genome rearrangements.
Busuttil
Rita A
RA
Buck Institute for Age Research, Novato, California, United States of America.
Muñoz
Denise P
DP
Garcia
Ana Maria
AM
Rodier
Francis
F
Kim
Woo Ho
WH
Suh
Yousin
Y
Hasty
Paul
P
Campisi
Judith
J
Vijg
Jan
J
D019588:Aging, Premature / Q000235:genetics*; D000818:Animals; D034961:Antigens, Nuclear; D002478:Cells, Cultured; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000172:deficiency*; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D042822:Genomic Instability; D007700:Kinetics; D000072200:Ku Autoantigen; D007763:Lac Operon / Q000235:genetics*; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D009928:Organ Specificity
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen